Over the past several years, we have been investigating the role of viral reservoirs in the pathogenesis of HIV disease in infected individuals. Despite development of highly active antiviral therapy (HAART) and its enormous success in the treatment of HIV-infected individuals, it has been clearly demonstrated that viral replication persists in the majority of infected individuals receiving HAART in whom plasma viremia has fallen below the limit of detection. In order to better understand the role of viral reservoirs in HIV infection, we have conducted the following two studies in the past year: 1) determination of the relationship between low levels of on-going viral replication and immunologic parameters, such as CD4+/CD8+ T cell ratios, in infected individuals in whom plasma viremia had been successfully suppressed by HAART and 2) examination of the range of the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in latently infected, resting CD4+ T cells in viremic versus aviremic individuals. In the first study, we have demonstrated a statistically significant inverse correlation between the frequency of CD4+ T cells carrying HIV-1 proviral DNA and the CD4+/CD8+ T cell ratios in aviremic infected individuals receiving HAART in whom plasma viremia had been suppressed below the limit of detection for prolonged periods of time (>2.5 years). No correlation was found between the frequency of HIV-1-specific cytotoxic CD8+ T lymphocytes (CTL) and the CD4+/CD8+ T cell ratios in those individuals. Our data suggest that persistent, low-level, on-going viral replication, although not sufficient to maintain HIV-1-specific CTL responses, may explain in part why normalization of the CD4+/CD8+ T cell ratios is not achieved in some infected individuals successfully treated with HAART. The presence of HIV-1 in latently infected, resting CD4+ T cells has been clearly demonstrated in infected individuals; however, the range of the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in this latent viral reservoir have not been fully delineated. In the second study, we have shown that highly purified resting CD4+ T cells from the majority of viremic patients whom we examined were capable of producing cell-free HIV-1 spontaneously ex vivo as measured by RT-PCR. The levels of HIV-1 released by resting CD4+ T cells were not significantly reduced in the presence of inhibitors of cellular proliferation and viral replication. In contrast, resting CD4+ T cells obtained from the majority of aviremic patients, which were phenotypically identical to those obtained from viremic patients, failed to produce cell-free HIV-1, despite levels of HIV-1 proviral DNA and cell-associated HIV-1 RNA comparable to those of viremic patients. DNA microarray analysis of resting CD4+ T cells demonstrated that a number of genes involving transcription regulation, RNA processing and modification, and protein trafficking and vesicle transport were significantly upregulated in resting CD4+ T cells from viremic patients compared to those of aviremic patients. These results suggest that active viral replication, as manifested by detectable plasma viremia, has a significant impact on the physiologic state of resting CD4+ T cells in infected patients, and in turn, allows release of cell-free HIV-1 without exogenous activation stimuli. In addition, given that no quantifiable virions were produced by the latent viral reservoir in the majority of aviremic patients despite the presence of cell-associated HIV-1 RNA, evidence for transcription of HIV-1 RNA in resting CD4+ T cells of aviremic patients should not necessarily be taken as direct evidence for ongoing viral replication during effective therapy.